Population genetics for 18 short tandem repeat loci (Canine GenotypesTM Panel 2.1 Kit) of 150 unrelated dogs from three pure-bred groups in Japan.

Similar to that in Europe and the United States, the need for forensic DNA identification in dogs is increasing in Japan. As few studies have used commercial genotyping kits, the effectiveness of the Canine GenotypesTM Panel 2.1 Kit for individual DNA identification in dogs bred in Japan was examined. We genotyped 150 unrelated dogs (50 Golden Retrievers, 50 Miniature Dachshunds, and 50 Shiba Inu) at 18 canine short tandem repeat loci by the Kit. The allele frequency, expected heterozygosity, observed heterozygosity, p-value, power of the discriminant, and of exclusion, polymorphic information content, and random matching probability were calculated for each marker. The random matching probability was subsequently estimated to be 4.394×10-22 in the 150 dogs of the three pure-bred groups based on 18 STR loci; 3.257 × 10-16 in the Golden Retriever, 3.933 × 10-18 in the Miniature Dachshund, and 2.107 × 10-18 in the Shiba Inu breeds. In addition, principal component analysis based on genotype data revealed the Golden Retrievers, Miniature Dachshunds, and Shiba Inus separated into three clusters. The results of the genotype analysis showed that the Canine GenotypesTM Panel 2.1 Kit could be useful for identity testing and tool of population study of canines in Japan.

Whole exome and transcriptome analysis revealed the activation of ERK and Akt signaling pathway in canine histiocytic sarcoma.

Histiocytic sarcoma (HS) is an incurable aggressive tumor, and no consensus has been made on the treatment due to its rare occurrence. Since dogs spontaneously develop the disease and several cell lines are available, they have been advocated as translational animal models. In the present study, therefore, we explored gene mutations and aberrant molecular pathways in canine HS by next generation sequencing to identify molecular targets for treatment. Whole exome sequencing and RNA-sequencing revealed gene mutations related to receptor tyrosine kinase pathways and activation of ERK1/2, PI3K-AKT, and STAT3 pathways. Analysis by quantitative PCR and immunohistochemistry revealed that fibroblast growth factor receptor 1 (FGFR1) is over-expressed. Moreover, activation of ERK and Akt signaling were confirmed in all HS cell lines, and FGFR1 inhibitors showed dose-dependent growth inhibitory effects in two of the twelve canine HS cell lines. The findings obtained in the present study indicated that ERK and Akt signaling were activated in canine HS and drugs targeting FGFR1 might be effective in part of the cases. The present study provides translational evidence that leads to establishment of novel therapeutic strategies targeting ERK and Akt signaling in HS patients.

Establishment of a BRAF V595E-mutant canine prostate cancer cell line and the antitumor effects of MEK inhibitors against canine prostate cancer.

Canine prostate cancer (cPCa) is a malignant neoplasm with no effective therapy. The BRAF V595E mutation, corresponding to the human BRAF V600E mutation, is found frequently in cPCa. Activating BRAF mutations are recognized as oncogenic drivers, and blockade of MAPK/ERK phosphorylation may be an effective therapeutic target against BRAF-mutated tumours. The aim of this study was to establish a novel cPCa cell line and to clarify the antitumor effects of MEK inhibitors on cPCa in vitro and in vivo. We established the novel CHP-2 cPCa cell line that was derived from the prostatic tissue of a cPCa patient. Sequencing of the canine BRAF gene in two cPCa cell lines revealed the presence of the BRAF V595E mutation. MEK inhibitors (trametinib, cobimetinib and mirdametinib) strongly suppressed cell proliferation in vitro, and trametinib showed the highest efficacy against cPCa cells with minimal cytotoxicity to non-cancer COPK cells. Furthermore, we orally administered 0.3 or 1.0 mg/kg trametinib to CHP-2 xenografted mice and examined its antitumor effects in vivo. Trametinib reduced tumour volume, decreased phosphorylated ERK levels, and lowered Ki-67 expression in xenografts in a dose-dependent manner. Although no clear adverse events were observed with administration, trametinib-treated xenografts showed osteogenesis that was independent of dosage. Our results indicate that trametinib induces cell cycle arrest by inhibiting ERK activation, resulting in cPCa tumour regression in a dose-dependent manner. MEK inhibitors, in addition to BRAF inhibitors, may be a targeted agent option for cPCa with the BRAF V595E mutation.

A feline case of multiple myeloma treated with bortezomib.

BACKGROUND: Multiple myeloma (MM) is an uncommon neoplasm in cats. There is no established standard of treatment due to the rare occurrence of this disease in cats. Bortezomib is a proteasome inhibitor that serves as the first-line drug for MM in humans, but its effectiveness currently is unknown in feline MM. We present here the case report of a feline MM that exhibited a favorable response to bortezomib. CASE PRESENTATION: The case was an 11-year-old non-castrated male domestic cat with light-chain MM presenting with clinical symptoms (anorexia, fatigue, and vomiting), mild azotemia, and pancytopenia. The cat failed on melphalan with prednisolone (MP), so bortezomib (Velcade) was initiated on Day 88. A total of 6 cycles of the treatment was performed, with each treatment cycle consisting of twice-weekly subcutaneous administration for 2 weeks followed by a 1-week rest. The dose of bortezomib was 0.7 mg/m2 for first week and 1.0 mg/m2 for second week in the first cycle. A dose of 0.7 mg/m2 was used for subsequent cycles. Prednisolone was used concomitantly in the first 2 cycles. Following treatment with bortezomib, clinical symptoms disappeared and a decrease in serum globulin and recovery of pancytopenia were noted. A monoclonal gammopathy, overproduction of serum immunoglobulin light chain, and Bence-Jones proteinuria that existed at diagnosis were undetectable on Day 123. A monoclonal gammopathy also was not detectable at the end of the bortezomib treatment (Day 213). Anorexia, fatigue, and marked bone marrow toxicity were experienced when bortezomib was administrated at a dose of 1.0 mg/m2, while no recognizable toxicity was observed at a dose of 0.7 mg/m2 throughout the treatment period. The case was placed on follow-up and there was no evidence of relapse as of Day 243. CONCLUSIONS: Bortezomib was effective and durable for the treatment of this case of feline MM after failure with MP. Bortezomib was well-tolerated in this cat at a dose of 0.7 mg/m2, but not at 1.0 mg/m2. Bortezomib appears to be a drug worthy of further study for the treatment of feline MM.

Molecular characterization of canine SHP2 mutants and anti-tumour effect of SHP2 inhibitor, SHP099, in a xenograft mouse model of canine histiocytic sarcoma.

Canine histiocytic sarcoma (HS) is an aggressive and highly metastatic neoplasm. Mutations in src homology 2 domain-containing phosphatase 2 (SHP2; encoded by PTPN11), which recently have been identified in canine HS tumour cells, could be attractive therapeutic targets for SHP099, an allosteric inhibitor of SHP2. Here, molecular characteristics of wild-type SHP2 and four SHP2 mutants (p.Ala72Gly, p.Glu76Gln, p.Glu76Ala and p.Gly503Val), including one that was newly identified in the present study, were investigated. Furthermore, in vivo effects of SHP099 on a HS cell line carrying SHP2 p.Glu76Ala were examined using a xenograft mouse model. While SHP2 Glu76 mutant cell lines and SHP2 wild-type/Gly503 mutant cell lines are highly susceptible and non-susceptible to SHP099, respectively, a cell line carrying the newly identified SHP2 p.Ala72Gly mutation exhibited moderate susceptibility to SHP099. Among recombinant wild-type protein and four mutant SHP2 proteins, three mutants (SHP2 p.Ala72Gly, p.Glu76Gln, p.Glu76Ala) were constitutively activated, while no activity was detected in wild-type SHP2 and SHP2 p.Gly503Val. Activities of these constitutively activated proteins were suppressed by SHP099; in particular, Glu76 mutants were highly sensitive. In the xenograft mouse model, SHP099 showed anti-tumour activity against a SHP2 p.Glu76Ala mutant cell line. Thus, there was heterogeneity in molecular characteristics among SHP2 mutants. SHP2 p.Glu76Ala and perhaps p.Glu76Gln, but not wild-type SHP2 or SHP2 p.Gly503Val, were considered to be oncogenic drivers targetable with SHP099 in canine HS. Further studies will be needed to elucidate the potential of SHP2 p.Ala72Gly as a therapeutic target of SHP099 in canine HS.

A canine case of malignant melanoma carrying a KIT c.1725_1733del mutation treated with toceranib: a case report and in vitro analysis.

BACKGROUND: Canine malignant melanoma is highly aggressive and generally chemoresistant. Toceranib is a kinase inhibitor drug that inhibits several tyrosine kinases including the proto-oncogene receptor tyrosine kinase KIT. Although canine malignant melanoma cells often express KIT, a therapeutic effect for toceranib has yet to be reported for this tumor, with only a small number of patients studied to date. This is a case report of a dog with malignant melanoma that experienced a transient response to toceranib. Furthermore, the KIT expressed in the tumor of this case was examined using molecular analysis. CASE PRESENTATION: A Shiba Inu dog presented with a gingival malignant melanoma extending into surrounding structures with metastasis to a submandibular lymph node. The dog was treated with toceranib (Palladia®; 2.6-2.9 mg/kg, orally, every other day) alone. Improvement of tumor-associated clinical signs (e.g., halitosis, tumor hemorrhage, trismus, and facial edema) with reduced size of the metastatic lymph node was observed on Day 15. The gingival tumor and associated masses in the masseter and pterygoid muscles decreased in size by Day 29 of treatment. Toceranib treatment was terminated on Day 43 due to disease progression and the dog died on Day 54. The tumor of this dog had a novel deletion mutation c.1725_1733del within KIT and the mutation caused ligand-independent phosphorylation of KIT, which was suppressed by toceranib. This mutation was considered to be an oncogenic driver mutation in the tumor of this dog, thereby explaining the anti-tumor activity of toceranib. CONCLUSIONS: This is the first report that presents a canine case of malignant melanoma that responded to toceranib therapy. KIT encoded by KIT harboring a mutation c.1725_1733del is a potential therapeutic target for toceranib in canine malignant melanoma. Further investigation of the KIT mutation status and toceranib therapy in canine malignant melanoma will need to be undertaken.

Commitment toward cell death by activation of autophagy with survivin inhibitor YM155 in two canine squamous cell carcinoma cell lines with high expression of survivin.

Canine squamous cell carcinoma (SCC) is difficult to treat if local therapy is not feasible. Recently, survivin inhibitor YM155 was shown to have growth inhibitory activity on high-survivin-expressing canine SCC cell lines HAPPY and SQ4. Here, the mechanisms underlying the effect of YM155 on these cell lines were investigated. YM155 induced cleavage of poly(ADP-ribose) polymerase (PARP) in HAPPY, but not in SQ4 cells. Analyzing two autophagy markers, the level of microtubule-associated protein 1 light chain 3 (LC3)-II and the LC3-II/LC3-I ratio, indicated that YM155 activates autophagy in both cell lines, and this activation occurs prior to PARP cleavage in HAPPY cells. Moreover, inhibition of autophagic flux by chloroquine almost completely prevented the toxic effect of YM155 in both cell lines. Although there are differences in their eventual cell death type, both cell lines may be committed to cell death by activation of autophagy with YM155. Activation of autophagy is likely to be a key mechanism in the growth-inhibitory effects of YM155 in these lines. These data provide new insights into the cytotoxic mechanism of YM155 in canine SCC cells.

Foxp3+ Regulatory T Cells Associated With CCL17/CCR4 Expression in Carcinomas of Dogs.

Regulatory T cells (Tregs) can be targeted in cancer immunotherapy. A previous study has shown that the chemokine CCL17 and the receptor CCR4 play a role in Treg recruitment in canine urothelial carcinoma. Here, we describe the association of tumor-infiltrating Tregs with CCL17/CCR4 expression in dogs with other carcinomas. In this study, we investigated 23 dogs with mammary carcinoma, 14 dogs with oral squamous cell carcinoma, 16 dogs with pulmonary adenocarcinoma, and 8 healthy control dogs. Immunohistochemistry showed that Foxp3+ Tregs and CCR4+ cells were increased in the tumor tissues of mammary carcinoma, squamous cell carcinoma, and pulmonary adenocarcinoma, when compared with the healthy tissues. The number of CCR4+ cells was associated with that of Foxp3+ Tregs. Double immunofluorescence labeling confirmed that most tumor-infiltrating Foxp3+ Tregs expressed CCR4. In vitro, canine carcinoma cell lines expressed CCL17 mRNA. Quantitative RT-PCR (reverse transcriptase-polymerase chain reaction) showed that CCL17 mRNA expression in canine carcinomas was increased approximately 10- to 25-fold relative to that of healthy tissues. These results suggest that the CCL17/CCR4 axis may drive Treg recruitment in a variety of canine carcinomas. CCR4 blockade may be a potential therapeutic option for tumor eradication through Treg depletion.

Nimustine Treatment of 11 Cases of Canine Histiocytic Sarcoma.

The objective of this retrospective study was to report treatment outcomes in dogs with histiocytic sarcoma (HS) that were treated with nimustine (ACNU). This study evaluated data from 11 dogs including 5 with macroscopic tumors that were treated in the primary setting and 6 that underwent aggressive local therapy while being treated in the adjuvant setting. The median ACNU starting dose was 25 mg/m2 (range, 20-30 mg/m2; 3- to 5-wk intervals, 1-8 administrations). The median overall survival in the primary and adjuvant settings was 120 days (median progression-free survival [PFS], 63 days) and 400 days (median PFS, 212 days), respectively. Neutropenia was observed in eight cases (grade 1, n = 1; grade 2, n = 2; grade 3, n = 2; grade 4, n = 3) with nadir neutrophil count at 1 wk after ACNU administration. Mild gastrointestinal toxicity (grade 1-2) was observed in three cases. ACNU was well tolerated and showed a similar outcome to that seen for lomustine, which is a drug commonly used to treat canine HS, in terms of overall survival and PFS in the current study population. Further investigations will need to be undertaken to definitively determine if ACNU is an appropriate alternative to lomustine for the treatment of HS.

The secondary KIT mutation p.Ala510Val in a cutaneous mast cell tumour carrying the activating mutation p.Asn508Ile confers resistance to masitinib in dogs.

BACKGROUND: Gain-of-function mutations in KIT are driver events of oncogenesis in mast cell tumours (MCTs) affecting companion animals. Somatic mutations of KIT determine the constitutive activation of the tyrosine kinase receptor leading to a worse prognosis and a shorter survival time than MCTs harbouring wild-type KIT. However, canine MCTs carrying KIT somatic mutations generally respond well to tyrosine kinase inhibitors; hence their presence represents a predictor of treatment effectiveness, and its detection allows implementing a stratified medical approach. Despite this, veterinary oncologists experience treatment failures, even with targeted therapies whose cause cannot be elucidated. The first case of an MCT-affected dog caused by a secondary mutation in the tyrosine kinase domain responsible for resistance has recently been reported. The knowledge of this and all the other mutations responsible for resistance would allow the effective bedside implementation of a deeply stratified and more effective medical approach. CASE PRESENTATION: The second case of a canine MCT carrying a different resistance mutation is herein described. The case was characterised by aggressive behaviour and early metastasis unresponsive to both vinblastine- and masitinib-based treatments. Molecular profiling of the tumoural masses revealed two different mutations; other than the already known activating mutation p.Asn508Ile in KIT exon 9, which is tyrosine kinase inhibitor-sensitive, a nearly adjacent secondary missense mutation, p.Ala510Val, which had never before been described, was detected. In vitro transfection experiments showed that the secondary mutation did not cause the constitutive activation by itself but played a role in conferring resistance to masitinib. CONCLUSIONS: This study highlighted the importance of the accurate molecular profiling of an MCT in order to improve understanding of the molecular mechanism underlying tumourigenesis and reveal chemoresistance in MCTs for more effective therapies. The detection of the somatic mutations responsible for resistance should be included in the molecular screening of MCTs, and a systematic analysis of all the cases characterised by unexpected refractoriness to therapies should be investigated in depth at both the genetic and the phenotypic level.

Canine histiocytic sarcoma cell lines with SHP2 p.Glu76Gln or p.Glu76Ala mutations are sensitive to allosteric SHP2 inhibitor SHP099.

Some canine cases of histiocytic sarcoma (HS) carry an activating mutation in the src homology two domain-containing phosphatase 2 (SHP2) encoded by PTPN11. SHP099 is an allosteric inhibitor of SHP2 that stabilizes SHP2 in a folded, auto-inhibited conformation. Here, we examined the expression and mutation status of SHP2 in five canine HS cell lines and evaluated the growth inhibitory properties of SHP099 against these cell lines. All five of the canine HS cell lines expressed SHP2, with three of the lines each harbouring a distinct mutation in PTPN11/SHP2 (p.Glu76Gln, p.Glu76Ala and p.Gly503Val). In silico analysis suggested that p.Glu76Gln and p.Glu76Ala, but not p.Gly503Val, promote shifting of the SHP2 conformation from folded to open-active state. SHP099 potently suppressed the growth of two of the mutant cell lines (harbouring SHP2 p.Glu76Gln or p.Glu76Ala) but not that of the other three cell lines. In addition, SHP099 suppressed ERK activation in the cell line harbouring the SHP2 p.Glu76Ala mutation. The SHP2 p.Glu76Gln and p.Glu76Ala mutations are considered to be activating mutations, and the signal from SHP2 p.Glu76Ala is inferred to be transduced primarily via the ERK pathway. Moreover, SHP099-sensitive HS cells, including those with SHP2 p.Glu76Gln or p.Glu76Ala mutations, may depend on these mutations for growth. Therefore, targeting cells harbouring SHP2 p.Glu76Gln and p.Glu76Ala with SHP099 may be an approach for the treatment of canine HS.

Genetic alterations of KIT during clonal expansion and subsequent acquisition of resistance to toceranib in a canine mast cell tumor cell line.

One of the potential mechanisms underlying acquired resistance to toceranib in canine mast cell tumor (MCT) is the emergence of a secondary mutation in the KIT gene. Here, genetic alterations of KIT during clonal expansion and subsequent acquisition of resistance to toceranib were investigated in the toceranib-susceptible canine MCT cell line VI-MC, which carries a KIT-activating mutation resulting in a predicted p.(Asn508Ile) amino acid change in the receptor tyrosine kinase protein KIT. Two sublines were cloned from VI-MC and toceranib-resistant sublines then were established by continuous exposure to toceranib. The mutation status of KIT in parental VI-MC and its sublines was investigated using next-generation sequencing (NGS). Additionally, effects of secondary mutations on toceranib sensitivity in p.(Asn508Ile)-mutant KIT were examined. KIT secondary mutations, including those encoding p.(Asn679Lys)-, p.(Asp819Val)-, and p.(Asp819Gly)-mutant KIT, that confer toceranib insensitivity to p.(Asn508Ile)-mutant KIT emerged only in toceranib-resistant VI-MCs. These mutations were not detected by NGS in the parental VI-MC line or in the toceranib-naive cloned VI-MCs, although the parental line and sublines exhibited genetic heterogeneity in KIT that may have been caused by genetic evolution during clonal expansion. VI-MC clones with these secondary mutations in KIT appear to have arisen from subclones during treatment with toceranib rather than being pre-existing. However, further study using a higher resolution technique will be needed to confirm the developmental mechanism of KIT secondary mutation in canine MCT cells with acquired resistance to toceranib.

Anti-tumour activity of oncolytic reovirus against canine histiocytic sarcoma cells.

Canine histiocytic sarcoma is an aggressive, fatal neoplastic disease with a poor prognosis. Lomustine is generally accepted as the first-line systemic therapy, although this compound does not provide complete regression. Therefore, research into a novel approach against canine histiocytic sarcoma is needed. However, anti-tumour effects of oncolytic therapy using reovirus against histiocytic sarcoma are unknown. Here, we showed that reovirus has oncolytic activity in canine histiocytic sarcoma cell lines in vitro and in vivo. We found that reovirus can replicate and induce caspase-dependent apoptosis in canine histiocytic sarcoma cell lines. A single intra-tumoural injection of reovirus completely suppressed the growth of subcutaneously grafted tumours in NOD/SCID mice. Additionally, we demonstrated that susceptibility to reovirus-induced cell death was attributable to the extent of expression of type I interferons induced by reovirus infection in vitro. In conclusion, oncolytic reovirus appears to be an effective treatment option for histiocytic sarcoma, and therefore warrants further investigation in early clinical trials.

Establishment and characterization of a cell line from a feline histiocytic sarcoma.

Feline histiocytic sarcoma (HS) is an aggressive and uncommon tumor originating from dendritic cells/macrophages. Here, a feline HS cell line, FHS-1, was established from a case of feline HS and characterized. Immunohistochemically, FHS-1 cells were positive for vimentin and Iba-1, and negative for MHC class II and CD163. FHS-1 cells were positive for α-naphthyl butyrate esterase staining, which was clearly inhibited by sodium fluoride. FHS-1 cells had phagocytic and antigen uptake/processing activities. Moreover, FHS-1 cells were tested for susceptibility to feline infectious peritonitis virus (FIPV) strain 79-1146; however, this cell line was not susceptible to this viral strain. Although FHS-1 cells lost the expression of MHC class II and CD163, our findings indicate that FHS-1 is a feline HS cell line that retains functional properties of dendritic cells/macrophages in terms of phagocytic and antigen uptake/processing activities. While FHS-1 cells are not suitable for in vitro study of FIP using strain 79-1146, they may be applicable for studies aimed at developing new diagnostic and therapeutic strategies for feline HS.

A decrease in ubiquitination and resulting prolonged life-span of KIT underlies the KIT overexpression-mediated imatinib resistance of KIT mutation-driven canine mast cell tumor cells.

Overexpression of KIT is one of the mechanisms that contributes to imatinib resistance in KIT mutation-driven tumors. Here, the mechanism underlying this overexpression of KIT was investigated using an imatinib-sensitive canine mast cell tumor (MCT) line CoMS, which has an activating mutation in KIT exon 11. A KIT-overexpressing imatinib-resistant subline, rCoMS1, was generated from CoMS cells by their continuous exposure to increasing concentrations of imatinib. Neither a secondary mutation nor upregulated transcription of KIT was detected in rCoMS1 cells. A decrease in KIT ubiquitination, a prolonged KIT life-span, and KIT overexpression were found in rCoMS1 cells. These events were suppressed by withdrawal of imatinib and were re-induced by re­treatment with imatinib. These findings suggest that imatinib elicited overexpression of KIT via suppression of its ubiquitination. These results also indicated that imatinib-induced overexpression of KIT in rCoMS1 cells was not a permanently acquired feature but was a reversible response of the cells. Moreover, the pan deubiquitinating enzyme inhibitor PR619 prevented imatinib induction of KIT overexpression, suggesting that the imatinib-induced decrease in KIT ubiquitination could be mediated by upregulation and/or activation of deubiquitinating enzyme(s). It may be possible that a similar mechanism of KIT overexpression underlies the acquisition of imatinib resistance in some human tumors that are driven by KIT mutation.

Identification of a secondary mutation in the KIT kinase domain correlated with imatinib-resistance in a canine mast cell tumor.

Imatinib-resistance is a major therapeutic problem in human chronic myeloid leukemia, human gastrointestinal stromal tumors, and canine mast cell tumors. In the present study, we identified the secondary mutation c.2006C>T in c-KIT exon 14 in a mast cell tumor obtained from a dog carrying c.1663-1671del in exon 11 and showing resistance to imatinib. The mutation in exon 14 resulted in substitution of threonine with isoleucine at position 669, which was located at the center of the ATP binding site as a gatekeeper and played an important role in binding to imatinib. Transfectants were constructed to survey the functions of these mutations in exons 11 and 14. The transfectant with mutant KIT encoded by c-KIT carrying c.1663-1671del showed constitutive ligand-independent phosphorylation that was suppressed by imatinib, indicating a gain-of-function mutation. Furthermore, the transfectant with mutant KIT encoded by c-KIT carrying both c.1663-1671del and c.2006C>T caused ligand-independent phosphorylation, which was not suppressed by imatinib. From these results, we concluded that the mutation c.2006C>T in c-KIT exon 14 was an imatinib-resistance mutation in a canine mast cell tumor. These findings revealed, for the first time, a mechanism of imatinib resistance in a clinical case of canine mast cell tumor.

Canine REIC/Dkk-3 interacts with SGTA and restores androgen receptor signalling in androgen-independent prostate cancer cell lines.

BACKGROUND: The pathological condition of canine prostate cancer resembles that of human androgen-independent prostate cancer. Both canine and human androgen receptor (AR) signalling are inhibited by overexpression of the dimerized co-chaperone small glutamine-rich tetratricopeptide repeat-containing protein α (SGTA), which is considered to cause the development of androgen-independency. Reduced expression in immortalised cells (REIC/Dkk-3) interferes with SGTA dimerization and rescues AR signalling. This study aimed to assess the effects of REIC/Dkk-3 and SGTA interactions on AR signalling in the canine androgen-independent prostate cancer cell line CHP-1. RESULTS: Mammalian two-hybrid and Halo-tagged pull-down assays showed that canine REIC/Dkk-3 interacted with SGTA and interfered with SGTA dimerization. Additionally, reporter assays revealed that canine REIC/Dkk-3 restored AR signalling in both human and canine androgen-independent prostate cancer cells. Therefore, we confirmed the interaction between canine SGTA and REIC/Dkk-3, as well as their role in AR signalling. CONCLUSIONS: Our results suggest that this interaction might contribute to the development of a novel strategy for androgen-independent prostate cancer treatment. Moreover, we established the canine androgen-independent prostate cancer model as a suitable animal model for the study of this type of treatment-refractory human cancer.

MicroRNA expression profiling in canine prostate cancer.

Canine prostate cancer (cPCa) is an untreatable malignant neoplasm resulting in local tissue invasion and distant metastasis. MicroRNAs (miRs) are small non-coding RNAs that function as oncogenes or tumor suppressors. The purpose of this study was to characterize the expression of miRs that are altered in cPCa tissue. The expression levels of 277 mature miRs in prostatic tissue (n=5, respectively) were compared between the non-tumor and tumor groups using real-time PCR. Five miRs (miR-18a, 95, 221, 222 and 330) were up-regulated, but 14 miRs (miR-127, 148a, 205, 299, 329b, 335, 376a, 376c, 379, 380, 381, 411, 487b and 495) were down-regulated specifically in cPCa (P<0.05). These miRs have potential use as early diagnosis markers for cPCa and in miR-based therapy.

Oncocytic carcinoma of the salivary gland in a dog.

A 3-y-old male miniature Dachshund was presented with an ~0.8 cm diameter mass in the right mandibular region. Fourteen months later, the mass was 5 × 4 × 3 cm. Grossly, the mass was encapsulated and was homogeneously gray-white on cut surface. Microscopically, the mass was composed of large, round to polygonal tumor cells that were arranged in solid nests and cords separated by a fibrovascular stroma. Tumor cells had large, round, hypochromatic nuclei containing large prominent nucleoli and abundant eosinophilic cytoplasm containing dark blue granules visible with phosphotungstic acid-hematoxylin stain. Metastasis was observed in the mandibular lymph node. Immunohistochemically, tumor cells were positive for CK AE1/AE3, low-molecular-weight CK (CAM5.2), E-cadherin, mitochondria ATPase beta subunit, and S100, but were negative for vimentin, carcinoembryonic antigen, p63, CK14, CD10, and chromogranin A. Ultrastructurally, tumor cells contained numerous mitochondria. Therefore, the tumor was diagnosed as an oncocytic carcinoma of the mandibular gland.

Molecular Characterization of the Cytidine Monophosphate-N-Acetylneuraminic Acid Hydroxylase (CMAH) Gene Associated with the Feline AB Blood Group System.

Cat's AB blood group system (blood types A, B, and AB) is of major importance in feline transfusion medicine. Type A and type B antigens are Neu5Gc and Neu5Ac, respectively, and the enzyme CMAH participating in the synthesis of Neu5Gc from Neu5Ac is associated with this cat blood group system. Rare type AB erythrocytes express both Neu5Gc and Neu5Ac. Cat serum contains naturally occurring antibodies against antigens occurring in the other blood types. To understand the molecular genetic basis of this blood group system, we investigated the distribution of AB blood group antigens, CMAH gene structure, mutation, diplotypes, and haplotypes of the cat CMAH genes. Blood-typing revealed that 734 of the cats analyzed type A (95.1%), 38 cats were type B (4.9%), and none were type AB. A family of three Ragdoll cats including two type AB cats and one type A was also used in this study. CMAH sequence analyses showed that the CMAH protein was generated from two mRNA isoforms differing in exon 1. Analyses of the nucleotide sequences of the 16 exons including the coding region of CMAH examined in the 34 type B cats and in the family of type AB cats carried the CMAH variants, and revealed multiple novel diplotypes comprising several polymorphisms. Haplotype inference, which was focused on non-synonymous SNPs revealed that eight haplotypes carried one to four mutations in CMAH, and all cats with type B (n = 34) and AB (n = 2) blood carried two alleles derived from the mutated CMAH gene. These results suggested that double haploids selected from multiple recessive alleles in the cat CMAH loci were highly associated with the expression of the Neu5Ac on erythrocyte membrane in types B and AB of the feline AB blood group system.